The N-Myc-responsive lncRNA MILIP promotes DNA double-strand break repair through non-homologous end joining

Significance Here, we report that the lncRNA MILIP is transcriptionally regulated by N-Myc and functions to promote DNA double-strand break repair in neuroblastoma cells. MILIP is distinguished from other lncRNAs implicated in DNA repair through its scaffolding role in Ku complex formation, a requisite step to initiate the nonhomologous end-joining pathway. Our findings substantiate the long-postulated role of N-Myc in regulating DNA repair in neuroblastoma cells and reveal the functional importance of MILIP in cell survival, proliferation, and resistance to genotoxic stress, with practical implications of MILIP targeting, alone and in combination with DNA-damaging therapeutics, for neuroblastoma treatment.

or anti-Ku70 Ab, followed by immunoblotting and RT-PCR analysis. Ten percent of the precipitates were reserved for analysis by immunoblotting and RT-PCR as input controls. All processes were under RNase free conditions.

Subcellular fractionation
Cells were incubated with hypotonic buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.15% Triton X-100, complete™, EDTA-free Protease Inhibitor Cocktail) and swollen on ice for 15 min. Samples were centrifuged for 3 min at 12,000 × g, and the supernatant collected as the cytoplasmic fraction. The pellets were rinsed once with cold PBS and nuclear proteins extracted using an equal volume of buffer B (20 mM Hepes pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5% Triton X-100, complete™, EDTA-free Protease Inhibitor Cocktail) on ice for 15 min. Cytoplasmic and nuclear fractions were centrifuged at 16,000 × g for 20 min to remove insoluble debris.

Immunofluorescence (IF)
Cells grown on coverslips were fixed for 10 min (4% formaldehyde) at room temperature, washed using PBS and then permeabilized using permeabilization buffer (0.1% Triton X-100 in PBS containing 10% BSA) before incubation overnight at 4°C with primary Ab. After washing with ice cold PBS three times, cells were incubated with Alexa Fluor 488-conjugated secondary Ab or CY3conjugated secondary Ab in the dark. After washing with permeabilization buffer, the coverslip was mounted using the Pro-Long TM Glass Antifade Mountant with NucBlue reagent (ThermoFisher Scientific, P36981). Photomicrographs were collected using epifluorescence microscopy (Leica SP8).

In situ hybridization (ISH)
ISH assays were performed using the RNAscope® 2.5 HD Detection Reagent-BROWN (Advanced Cell Diagnostics, #322310) according to the manufacturer's instructions. Briefly, FFPE tissue microarrays were deparaffinized in xylene for 5 min at RT twice, followed by dehybridization in 100% alcohol. After being air-dried, the tissue sections were incubated with hydrogen peroxide for 10 min at RT and washed in the distilled water five times. Then the sections were heated in target retrieval reagent to 100 °C for 20 min, followed by treatment with proteinase K and incubated in hybridization buffer. containing probes at 40 °C for 3 h. After being washed, the sections were incubated with 3,3′-diaminobenzidine (DAB), and counterstaining was carried out using hematoxylin. The percentage of positive cells was ranged from 0 to 100%. The intensity of staining (intensity score) was judged on an arbitrary scale of 0-4: no staining (0), weakly positive staining (1), moderately positive staining (2), strongly positive staining (3) and very strong positive staining (4). A reactive score (RS) was derived by multiplying the percentage of positive cells with staining intensity divided by 10. Two researchers who were blinded to the information of FFPE tissue microarrays examined the ISH slides independently. The RS of each tissue was presented as the average of scores derived by the two researchers.

Immunohistochemistry (IHC)
FFPE tissue microarrays were deparaffinized in xylene for 5 min at RT twice, followed by dehybridization in 100% alcohol. Antigen retrieval was performed in a pressure cooker for 20 min in 10 mM Tris with 1 mM EDTA (pH 9). Endogenous peroxidase activity was inhibited with 1.5% H2O2 in methanol for 20 min followed by washing in PBS. Nonspecific binding was blocked using blocking buffer (PBS (pH 7.4), 3% serum, 1% BSA and 0.1% Tween) for 60 min at room temperature. Sections were then incubated with an anti-Ki67 antibody (Proteintech Group, 27309-1-AP, Wuhan, China) that were diluted in blocking buffer overnight at 4 °C. After washing twice with 0.1% PBS-Tween, slides were incubated with a secondary antibody (BOSTER Biological Technology, BM3894, Wuhan, China). After washing, sections were incubated with with 3,3′diaminobenzidine (DAB) (Sigma-Aldrich) followed by counterstaining with hematoxylin (Servicebio, G1004, Wuhan, China). After dehydration, sections were mounted using Permount TM Mounting Medium (Servicebio, WG10004160, Wuhan, China). Slides were examined by two investigators. The percentage of positive cells was estimated from 0% to 100%. The intensity of staining (intensity score) was judged on an arbitrary scale of 0-4: no staining (0), weakly positive staining (1), moderately positive staining (2), strongly positive staining (3), and very strongly positive staining (4). An immunoreactive score (IRS) was derived by multiplying the percentage of positive cells with staining intensity divided by 10. Two researchers who were blinded to the information of FFPE tissue microarrays examined the IHC slides independently. The IRS of each tissue was presented as the average of scores derived by the two researchers.

Comet assays
The CometAssay Single Cell Gel Electrophoresis Assay Kit was used according to manufacturer's instructions (Trevigen, 4250-050-K). Briefly, 500 cells (1 × 10 5 cells/ml) were mixed with lowmelting-point agarose on slides at 37 °C. After solidifying for 10 min at 4 °C, the slides were immersed in the lysis solution and then in freshly prepared alkaline unwinding solution to permit DNA unfolding followed by electrophoresis (21 V for 30 min). The slides were washed with double distilled H2O twice, immersed in 75% ethanol for 5 min, stained with PI. The percentage of tail DNA content of the comet was measured with Comet Assay IV software (Perceptive Instruments).

CRISPR/Cas9 knockout of p53
Single-guide RNA (sgRNA) sequences targeting p53 (5'-GGGCAGCTACGGTTTCCGTC-3') were cloned into the lentiCRISPR v2 plasmid (#52961, Addgene, Cambridge, MA, USA) by simple annealing and ligation. CHP-134 cells were transfected with the sgRNA construct using Lipofectamine 3000 reagent (ThermoFisher Scientific). Twenty-four hrs later, cells were subjected to selection in the culture medium containing puromycin (#A1113803, ThermoFisher Scientific) for 1 week. Viable cells were trypsinized, washed with the culture medium, and re-plated on 96 well plates in single cell suspension by limiting dilution and cultured for 12-14 days. Single cell colonies were picked, expanded, and assayed for p53 expression using Western blot analysis. The DAN indels in the TP53 locus of the two p53 knockout sublines (CHP-134.p53KO1 and CHP-134.p53KO2) were further confirmed by Sanger sequencing.

Quantitative PCR (qPCR)
Total RNA was extracted from cultured cells using the Gene JET RNA Purification Kit (ThermoFisher Scientific, #K0731) according to the manufacturer's instructions. cDNA was synthesized from 1 μg of total RNA using the PrimScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, #RR047A; Dalian, China). Of the resultant cDNA, 12.5 ng was used in the 20 μl qPCR mix, containing 10 μl of TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, #RR820A; Dalian, China) and 0.4 μM of each primer. Samples were amplified for 40 cycles using a StepOnePlusTM Real-Time PCR System (ThermoFisher Scientific). The 2-ΔΔCT method was used to calculate expression levels relative to the GAPDH or 18S rRNA housekeeping controls.

Luciferase reporter assays
The Dual-Luciferase® Reporter Assay System was performed according to manufacturer's instructions (Promega, E1910). Cells were transfected with the pGL3-based constructs containing MILIP promoter together with Renilla luciferase plasmids. Twenty-four hours later, Firefly and Renilla luciferase activities were examined by Varioskan LUX microplate reader (ThermoFisher). Renilla luciferase activities were used to normalize the firefly luciferase activity.

DSB repair reporter assays
The activity of DSB pathways was measured using GFP reporters for total NHEJ (EJ5-GFP; Addgene, 44026) and HR (DR-GFP; Addgene, 26475). Cells were co-transfected with MILIP or control siRNA, the reporter EJ5-GFP or DR-GFP, and a plasmid encoding the I-SceI endonuclease (Addgene, 26477) to introduce a DSB at I-SceI sites in the reporter constructs. GFP was measured using a flow cytometer (FACSCanto, BD Biosciences). A vector expressing GFP only was cotransfected in parallel to measure transfection efficiency. The percentage of GFP positive cells were normalized to transfection efficiency.

Absolute quantification of RNA
Absolute RNA quantification was performed using the standard curve method by qPCR. cDNA was prepared from a fixed cell number using the qScript cDNA SuperMix (Quantabio, Cat# 95048-500) in a 20 μL reaction and subsequently diluted to 100 μL. Ten-fold serial dilutions of the pcDNA3.1-MILIP plasmid (10 2 -10 7 molecules per ml) were used as a reference molecule for the standard curve calculation. Assays were reconstituted to a final volume of 20 μl using 5 μl cDNA from cells or 5 μl serial diluted pcDNA3.1-MILIP plasmid and cycled using a StepOnePlusTM Real-Time PCR System. Data calculated as copies per 5 μl cDNA were converted to copies per cell based on the known input cell equivalents. The sequences for primers are provided in Table S6.

Western blotting
Cells were lysed in lysis buffer (50mM Tris-HCl [pH 7.5], 150mM NaCl, 2.5mM MgCl2, 1mM EDTA, 10% Glycerol, 1% triton-100, 1mM DTT, complete™ EDTA-free protease inhibitor cocktail [Sigma-Aldrich, 4693132001]) and sonicated. Supernatant was collected after the samples were centrifugated at 13,000 × g at 4 °C for 20 min. Protein concentrations in the supernatant samples were quantified with the Bicinchoninic Acid Assay kit (Pierce, Rockford, IL). Equal amounts of protein samples were loaded onto each lane of sodium dodecyl sulfatepolyacrylamide (SDS) gel, followed by electrophoresis and transfer to nitrocellulose membranes. The membranes were blocked of nonspecific antibody binding with 10% skim milk powder in phosphate-buffered saline and probed with the primary antibody. After washing with TTBS, the membrane was then incubated with a horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibody. Protein bands were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce, 34079). Semi-quantitation of protein bands was carried out using the NIH ImageJ. The information of antibodies used is provided in Table S4.

Chromatin immunoprecipitation (ChIP)
ChIP assays were performed using the ChIP Assay Kit (Beyotime, #P2078; Shanghai, China) according to the manufacturer's instructions. Briefly, cells were cross-linked with a final concentration of 1% formaldehyde in growth medium for 15 min at 37 °C and quenched by the addition of glycine solution for 5 min at room temperature (RT). Then cells were harvested, lysed using cell lysis buffer (2% SDS [w/v], 150 mM NaCl, 50 mM Tris/HCl, 50 mM EDTA, pH 8.0) and sonicated. After being cleared by centrifugation at 12,000 × g for 10 min at 4 °C, the cell lysate was subjected to a 1:10 dilution and rotated with antibodies or corresponding mouse/rabbit normal immunoglobulin at 4 °C overnight. Then, 60 μl of protein A/G agarose beads was added to the antibody-lysate mixture and rotated at 4 °C for an additional 1 h. Beads were washed using the lysis buffer, and DNA fragments were eluted, purified and subjected to PCR analysis using the specific primers. PCR products were separated by gel electrophoresis on the 2% agarose gel. The sequences for primers are provided in Table S6.

RNA immunoprecipitation (RIP)
The EZ-Magna RIP Kit (Millipore, 17-701) was used according to manufacturer's instructions. Briefly, whole cell lysates prepared by ultrasonication in lysis buffer (50mM Tris-HCl [pH 7.5], 150mM NaCl, 2.5mM MgCl2, 1mM EDTA, 10% Glycerol, 1% triton-100, 1mM DTT, complete™ EDTA-free protease inhibitor cocktail [Sigma, 4693132001] and RiboLock RNase inhibitor [Life Technologies, EO0382]) were incubated with magnetic beads coated with the indicated Abs at 4 °C. After washing with lysis buffer 5 times, the bead-bound immunocomplexes were treated with proteinase K. Samples were then centrifuged and placed on a magnetic separator. Supernatants were used to extract RNA with a FastPure® Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, RC112-01) before subjecting the purified RNAs to PCR analysis. The sequences for primers are provided in Table S6.

Mass spectrometry (MS) analysis.
Cell lysates were prepared by ultrasonication in lysis buffer (50mM Tris-HCl [pH 7.5], 150mM NaCl, 2.5mM MgCl2, 1mM EDTA, 10% Glycerol, 1% triton-100, 1mM DTT, complete™ EDTA-free protease inhibitor cocktail [Sigma, 4693132001] and RiboLock RNase inhibitor [Life Technologies, EO0382]) followed by incubation with probes before rotating with streptavidin beads (ThermoFisher, 20349) for 2 hrs. The beads were washed in the lysis buffer five times and the retrieved proteins were separated by 12.5% SDS PAGE. Protein bands were visualized by Coomassie blue staining. Two bands at ~70 kDa and ~80 kDa, respectively, were observed in samples derived with MILIP antisense probes but not in samples derived with MILIP sense probes. The segment of the SDS gel corresponding to each band was cut out and processed for MS analysis. Peptides were sequenced by nanoflow reversed phased Liquid Chromatography (Dionex Ultimate 3000 RSLCnano, Dionex, Idstein, Germany) coupled directly to an ESI 3D Ion Trap Mass Spectrometer (Luming Biotechnology, Shanghai, China) operating in MS/MS (CID) mode (n = 1 technical replicate). Peptides were loaded at 5 μl/min onto a 5 μm C18 nanoViper trap column (100 μm × 2 cm, Acclaim PepMap100, Thermo) for desalting and pre-concentration. Peptide separation was then performed at 300 nl/min over an Acclaim nanoViper analytical column (2 μm C18, 75 μm × 15 cm) utilising a gradient of 2-40% Buffer B (80% Acetonitrile, 0.1% Formic Acid) over 60 min. The peptides were eluted directly into the nanoflow ESI Ion source of the MS system for MS/MS analysis. The AmaZon Ion Trap system was tuned using Smart Parameter Settings tuned to 922 m/z and set to perform MS/MS on the top 6 ions present in each MS scan with an Ion exclusion time of 30 sec. Source settings were as follows: dry gas temperature, 180 °C; dry gas, 4.0 L min−1; nebulizer gas, 0.4 bar; electrospray voltage, 4500 V; high-voltage endplate offset, -200 V; capillary exit, 140 V; trap drive, 57.4; funnel 1 in 100 V, out 35 V, and funnel 2 in 12 V, out 3.3 V; MS/MS ICC target 500,000; maximum accumulation time, 50 ms. The sample was measured with the Ultrascan Scan Mode in polarity positive, scan range from m/z 100-3000, 3 MSn spectra averages. MS/MS spectra were triggered on ions higher than 50,000 in the Profile scan using a fragmentation amplitude of 100%.
Raw MS Files were converted into MASCOT Generic Format using DataAnalysis 4.1 and imported into ProteinScape 2.1 platform (both Bruker, Bremen, Germany) for database searching. Searches were performed against the UniProt Swiss-Prot Human database (retrieved January 2017) using an in-house licensed MASCOT server (version 2.3.02, Matrix Science). The number of allowed trypsin missed cleavages set to 2. Deamidation of Asparagine and Glutamine, Oxidation of Methionine and Phosphorylation of Serine, Threonine and Tyrosine were set as variable modifications. The parent ion tolerance was set to 1.2 Da with fragment ion tolerance set to 0.7 Da. Peptide thresholds were set requiring False Positive Rate less than 0.05% with a low stringency MASCOT score greater than 35. Those spectra meeting these criteria were validated by manual inspection to ensure accurate y-and b-ion detection with overlapping sequence coverage.

In vitro transcription
The plasmid pcDNA3.1-MILIP was constructed by TSINGKE Biological Technology (Beijing, China). The plasmids were linearized by restriction enzyme BstBI (New England biolab, R0519S) and in vitro transcription was then performed using TranscriptAid T7 High Yield Transcription Kit (ThermoFisher Scientific, K0441) according to the manufacturer's instructions.

Xenograft mouse model
Cells (5 × 10 6 ) were subcutaneously injected into the dorsal flanks of 4-week-old female nu/nu mice (GemPharmatech, Shanghai, China) (6 mice per group), Treatment protocols are detailed in Table  S8. At the end of experiments, mice were sacrificed, and tumors excised and measured. Studies on animals were approved by the Animal Research Ethics Committee of the Academy of Medical Science of Zhengzhou University. In vivo treatment protocols were detailed in Table S8.

Statistical Analysis
Analysis was carried out using GraphPad Prism to assess differences between experimental groups. Statistical differences were analyzed by two-tailed Student's t-test or One-way ANOVA test followed by Tukey's multiple comparisons. P values lower than 0.05 were considered statistically significant.

Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) with the dataset identifier PXD033372. All other data supporting the findings of this study are available from the corresponding authors on request.  in ENCODE (Encyclopedia of DNA Elements). F, siRNA knockdown of N-Myc (as shown in Figure  1D) downregulated the expression of the lncRNA PVT1 (Data shown are mean ± SEM of 3 independent experiments. One-way ANOVA followed by Tukey's multiple comparison). G, N-Myc overexpression upregulated MILIP in SK-N-FI and SK-N-AS cells (Data shown are mean ± SEM of 3 independent experiments. Two-tailed Student's t-test). H, the transcriptional activity of a MILIP reporter construct containing the Myc-BR was increased by overexpression of N-Myc, whereas a MILIP reporter construct with the Myc-BR deleted displayed decreased activity that was not further affected by N-Myc overexpression (Data shown are mean ± SEM of 3 independent experiments. One-way ANOVA followed by Tukey's multiple comparison). I and J, neither knockdown (I) nor overexpression (J) of MILIP altered the expression of N-Myc as measured using immunoblotting (Data shown are mean ± SEM of 3 independent experiments. One-way ANOVA followed by Tukey's multiple comparison (I) or two-tailed Student's t-test (J).  Figure 2J and 2K showing that treatment with Dox (2 mg/ml supplemented with 10 mg/ml sucrose in drinking water) caused reductions in BE(2)-C.shMILIP.2 xenograft wights in nu/nu mice that was however reversed by the cessation of Dox treatment (n = 6 mice per group, mean ± SEM, one-way ANOVA followed by Tukey's multiple comparison). G, qPCR analysis of randomly selected BE(2)-C.shMILIP.2 tumor tissues harvested from nu/nu mice with or without cessation of Dox treatment showing MILIP expression levels (n = 3 tumors). One-way ANOVA followed by Tukey's multiple comparison). H, representative microscopic photographs of TUNEL staining on FFPE tissue sections from representative BE(2)-C.shMILIP.2 xenografts harvested from mice with or without Dox treatment to induce knockdown of MILIP (n = 3 tumors per group). I, quantitation of TUNEL positive cells as shown in H (n = 3 tumors per group. Two-tailed Student's t-test). J, representative microscopic photographs of IHC staining of Ki67 on FFPE tissue sections from representative BE(2)-C.shMILIP.2 xenografts harvested from mice with or without Dox treatment to induce knockdown     Figure 5E and F showing co-treatment with Dox (1 mg/ml supplemented with 10 mg/ml sucrose in drinking water) and CDDP (1mg/kg, i.p. injection) induced greater inhibition of BE(2)-C.shMILIP.2 xenograft growth than treatment with Dox or CCDP alone in nu/nu mice (n = 6 mice per group, mean ± SEM, one-way ANOVA followed by Tukey's multiple comparison).